Infusion of Red Blood Cell-Derived Microparticles (RMPs) Reduced Intracerebral Bleeding Following Hemorrhagic Stroke in a Rat Model

INTRODUCTION: Spontaneous intracerebral hemorrhage (SICH) is associated with significant morbidity and mortality, yet no effective treatment for it exists. It has been shown that the size of hematoma plays a major role in determining the outcomes of SICH. Therefore, the paradigm that reduction of hematoma growth, and/or prevention of continued bleeding in SICH has been an attractive therapeutic target. Various hemostatic agents including NovoSeven^® have previously been investigated for SICH, without benefit. Red blood cell-derived microparticles (RMP) have diverse hemostatic activity. It has been shown in vitro that RMP enhance platelet functions as well as accelerating coagulation, augmenting both primary and secondary hemostasis. Efficacy of RMP as hemostatic agent has been documented in animal bleeding models [Jy et al, Thrombosis & Haemostais, 2013; 110:751-60]. The present report provides preliminary data on efficacy of RMP infusion for reducing hematoma expansion in a rat model of SICH.

METHODS: RMP were produced by high pressure extrusion of washed RBC as described [Jy et al, Thrombosis & Haemostais, 2013; 110:751-60]. SICH was induced in male Sprague Dawley rats by injection of 0.17U of collagenase (2 µl at the rate of 0.4 µl / min) into the left striatum sterotexically. It has been shown that after injection of collagenase, intracerebral bleeding continued for several hours [Del Bigio et al. Stroke, 1996; 27:2312-9]. Rats were randomly divided into two groups (1) Placebo (vehicle-treated) group, n=9; and (2) RMP-treated group, n=10. RMP were injected via femoral vein at 1 hour and again at 2 hours post-SICH, at concentration 3x10⁹particles / kg, per injection. Rats were sacrificed at ~24 hours post-collagenase injection, and brains were sectioned. Sectioned brains were scanned and hematoma area on brain section images was evaluated by Image J software. The neurological score was evaluated at ~24 hr as described earlier [Bederson et al. Stroke. 1986; 17:472-476; De Ryck et al. Stroke. 1989; 20:1383-1390]. Induction of SICH and evaluation of hematoma area and neurological score was carried out by an investigator blinded to the experimental conditions.

RESULTS: (i)Effect of RMP treatment on hematoma area: We observed that total hematoma area for the placebo group was 105 ± 13 mm² (mean ±SEM) at ~24 hr post-collagenase injection. In contrast, in the RMP-treated group, total hematoma area was 72 ± 9 mm², which was 31% lower than in the placebo group; p <0.05. (ii)Mortality and morbidity. At ~24 hr post-SICH, one rat died in vehicle treated group while no mortality was observed in RMP group. We did not observe any significant difference in neurological score at ~24ht post-SICH. We believe that a longer follow-up (7 days) may yield significant differences, since it is known that ultimate outcomes of this injury, including death and overt CNS impairment, require several days to be manifest.

CONCLUSIONS: These preliminary results are promising, but indicate a need for longer-term observation and larger sample size. Modified dosage regimen might also improve efficacy since we have observed in other studies that continuous infusion of RMPs is more effective than bolus. In summary, results demonstrate that RMPs have the potential to arrest or limit hematoma growth in SICH. If further work confirms and improves this benefit, RMP treatment could save many lives.